LOCUS       40924_34744        7761 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPKm-105, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7761)
  AUTHORS   Kyriakakis P, Catanho M, Hoffner N, Thavarajah W, Jian-Yu V, Chao 
            SS, Hsu A, Pham V, Naghavian L, Dozier LE, Patrick G, Coleman T.
  TITLE     Biosynthesis of Orthogonal Molecules Using Ferredoxin and 
            Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB 
            Optogenetics
  JOURNAL   ACS Synth Biol (2018) In press
  PUBMED    29301067
REFERENCE   2  (bases 1 to 7761)
  AUTHORS   Kyriakakis P.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, 
            PFBH room 251 MC0412, La Jolla, CA 92093, United States of America
REFERENCE   3  (bases 1 to 7761)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7761)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth 
            Biol (2018) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-NOV-2017) Bioengineering, UCSD, 9500 Gilman drive, PFBH room 251
            MC0412, La Jolla, CA 92093, United States of America"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7761
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        27..406
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        407..610
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     promoter        655..673
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    696..2558
                     /label=PhyB 1-621
                     /note="PhyB 1-621"
     CDS             2571..2591
                     /codon_start=1
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /translation="PKKKRKV"
     CDS             2601..3041
                     /codon_start=1
                     /label=GAL4 DNA binding domain
                     /note="DNA binding domain of the GAL4 transcriptional
                     activator"
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3057..3083
                     /codon_start=1
                     /label=HA
                     /note="HA (human influenza hemagglutinin) epitope tag"
                     /translation="YPYDVPDYA"
     promoter        complement(3105..3123)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     polyA_signal    3149..3373
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3419..3847
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        3861..4191
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             4258..5049
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    5226..5359
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5396..5412)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5420..5436)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(5444..5474)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5489..5510)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5798..6386)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6560..7417)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7418..7522)
                     /label=AmpR promoter
ORIGIN
        1 cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc
       61 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
      121 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
      181 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg
      241 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      301 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
      361 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
      421 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
      481 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
      541 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
      601 aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg aaattaatac
      661 gactcactat agggagaccc aagcttggta ccaacatggt ttccggagtc gggggtagtg
      721 gcggtggccg tggcggtggc cgtggcggag aagaagaacc gtcgtcaagt cacactccta
      781 ataaccgaag aggaggagaa caagctcaat cgtcgggaac gaaatctctc agaccaagaa
      841 gcaacactga atcaatgagc aaagcaattc aacagtacac cgtcgacgca agactccacg
      901 ccgttttcga acaatccggc gaatcaggga aatcattcga ctactcacaa tcactcaaaa
      961 cgacgacgta cggttcctct gtacctgagc aacagatcac agcttatctc tctcgaatcc
     1021 agcgaggtgg ttacattcag cctttcggat gtatgatcgc cgtcgatgaa tccagtttcc
     1081 ggatcatcgg ttacagtgaa aacgccagag aaatgttagg gattatgcct caatctgttc
     1141 ctactcttga gaaacctgag attctagcta tgggaactga tgtgagatct ttgttcactt
     1201 cttcgagctc gattctactc gagcgtgctt tcgttgctcg agagattacc ttgttaaatc
     1261 cggtttggat ccattccaag aatactggta aaccgtttta cgccattctt cataggattg
     1321 atgttggtgt tgttattgat ttagagccag ctagaactga agatcctgcg ctttctattg
     1381 ctggtgctgt tcaatcgcag aaactcgcgg ttcgtgcgat ttctcagtta caggctcttc
     1441 ctggtggaga tattaagctt ttgtgtgaca ctgtcgtgga aagtgtgagg gacttgactg
     1501 gttatgatcg tgttatggtt tataagtttc atgaagatga gcatggagaa gttgtagctg
     1561 agagtaaacg agacgattta gagccttata ttggactgca ttatcctgct actgatattc
     1621 ctcaagcgtc aaggttcttg tttaagcaga accgtgtccg aatgatagta gattgcaatg
     1681 ccacacctgt tcttgtggtc caggacgata ggctaactca gtctatgtgc ttggttggtt
     1741 ctactcttag ggctcctcat ggttgtcact ctcagtatat ggctaacatg ggatctattg
     1801 cgtctttagc aatggcggtt ataatcaatg gaaatgaaga tgatgggagc aatgtagcta
     1861 gtggaagaag ctcgatgagg ctttggggtt tggttgtttg ccatcacact tcttctcgct
     1921 gcataccgtt tccgctaagg tatgcttgtg agtttttgat gcaggctttc ggtttacagt
     1981 taaacatgga attgcagtta gctttgcaaa tgtcagagaa acgcgttttg agaacgcaga
     2041 cactgttatg tgatatgctt ctgcgtgact cgcctgctgg aattgttaca cagagtccca
     2101 gtatcatgga cttagtgaaa tgtgacggtg cagcatttct ttaccacggg aagtattacc
     2161 cgttgggtgt tgctcctagt gaagttcaga taaaagatgt tgtggagtgg ttgcttgcga
     2221 atcatgcgga ttcaaccgga ttaagcactg atagtttagg cgatgcgggg tatcccggtg
     2281 cagctgcgtt aggggatgct gtgtgcggta tggcagttgc atatatcaca aaaagagact
     2341 ttcttttttg gtttcgatct cacactgcga aagaaatcaa atggggaggc gctaagcatc
     2401 atccggagga taaagatgat gggcaacgaa tgcatcctcg ttcgtccttt caggcttttc
     2461 ttgaagttgt taagagccgg agtcagccat gggaaactgc ggaaatggat gcgattcact
     2521 cgctccagct tattctgaga gactctttta aagaatcttt gcggccgcaa cctaagaaaa
     2581 agagaaaggt gggcggaggc atgaagctac tgtcttctat cgaacaagca tgcgatattt
     2641 gccgacttaa aaagctcaag tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga
     2701 acaactggga gtgtcgctac tctcccaaaa ccaaaaggtc tccgctgact agggcacatc
     2761 tgacagaagt ggaatcaagg ctagaaagac tggaacagct atttctactg atttttcctc
     2821 gagaagacct tgacatgatt ttgaaaatgg attctttaca ggatataaaa gcattgttaa
     2881 caggattatt tgtacaagat aatgtgaata aagatgccgt cacagataga ttggcttcag
     2941 tggagactga tatgcctcta acattgagac agcatagaat aagtgcgaca tcatcatcgg
     3001 aagagagtag taacaaaggt caaagacagt tgactgtatc gccggaattc atggcttacc
     3061 catacgatgt tccagattac gcttgataat ctagagggcc ctattctata gtgtcaccta
     3121 aatgctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     3181 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     3241 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     3301 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     3361 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     3421 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3481 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3541 ttcgccggct ttccccgtca agctctaaat cggggcatcc ctttagggtt ccgatttagt
     3601 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     3661 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     3721 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     3781 gggattttgg ggatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     3841 gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     3901 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3961 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4021 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4081 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4141 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     4201 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     4261 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4321 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4381 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     4441 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4501 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4561 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4621 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4681 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4741 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4801 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4861 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4921 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4981 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5041 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5101 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5161 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5221 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     5281 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     5341 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     5401 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5461 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5521 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5581 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5641 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5701 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5761 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5821 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5881 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5941 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac
     6001 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6061 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6121 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6181 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     6241 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     6301 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6361 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6421 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6481 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6541 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6601 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6661 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6721 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6781 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6841 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6901 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6961 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7021 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7081 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7141 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7201 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     7261 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     7321 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     7381 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7441 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7501 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     7561 cgggagatct cccgatcccc tatggtcgac tctcagtaca atctgctctg atgccgcata
     7621 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     7681 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     7741 taggcgtttt gcgctgcttc g
//
